1. Field of the Invention
The present invention relates to methods, compositions and kits for performing immunoassays to detect an immunologically non-remarkable compound, such as benzene.
2. Discussion of the Background
Testing is an essential, and integral, component of all environmental protection and restoration activities. It is the rate limiting element that influences the time, cost, and overall efficiency of project management.
The Environmental Protection Agency (EPA) has long promoted and supported the concept of screening methods to supplement laboratory analysis and increase overall efficiency. The need for more effective methods has been recognized in the Superfund Amendments and Reauthorization Act of 1986 which specifies the development and evaluation of alternative time and cost-saving methods that will assist in the eventual remediation of the nations Superfund sites.
Effective field screening methods can increase the efficiency of site management and improve overall data quality when used to supplement the services of regional laboratories. The development of these methods, however, requires a technology that will be compatible with numerous compounds and matrixes and yet be simple, effective and rugged enough to be incorporated into a protocol for use in the field.
Screening methods need to provide fast, simple, cost-effective and reliable information when operated under field conditions. The reagents and equipment should be portable and stable at ambient conditions, and the claims relating to performance should accurately reflect anticipated field use. The methods should be able to rapidly provide an ample quantity of data, and the protocol should be simple to perform and safe to use. Performance characteristics relative to sensitivity, freedom from matrix interferences and cross-reacting compounds, and correlation to an acceptable reference method should be carefully evaluated. Developers must maintain high, and consistent, quality standards relative to the consistency of their manufacturing protocols, the adequacy of in-process and pre-release quality control methods, and the reliability of their product claims. A necessary characteristic of particular significance for screening methods, is that they exhibit a very low frequency of false negative results.
Screening methods detect contamination at specified concentrations. The concentration may relate to a hazardous threshold, a clean-up target, or a process-control parameter. The potential implications of false negative data far outweigh those of false positive results. The consequence of a false positive, while a costly problem that needs to be minimized, results in additional testing or treatment. False negative data, however, provides an erroneous perception of a clean site, and may have serious environmental and legal consequences. Safeguards that minimize the incidence of false negative results are imperative. Appropriate control over the frequency of false positive data needs to be established and maintained.
Immunoassay methods combine the specific binding characteristics of an antibody molecule with a read-out system that is used to detect and quantify compounds. Current immunoassay technology benefits from the diversity of detection systems developed that use enzyme-catalyzed chromogenic reactions, radionuclides, chemiluminescence, fluorescence, fluorescence polarization and a variety of potentiometric and optical biosensor techniques. Improvements in the sensitivity achieved has necessitated the generation of new descriptive nomenclature for methods that can now detect "zeptomole" (10.sup.-21 moles=600 molecules) concentrations.
The U.S. Environmental Protection Agency has concluded that immunoassay technology provides an advantage over the traditional analytical methods (gas chromatography (GC), gas chromatography/mass spectrometry (GC/MS), high performance liquid chromatography (HPLC) by increasing the sample throughput at a lower cost (Van Emon et al. in Field Screening Methods for Hazardous Wastes and Toxic Chemicals, p. 815-818 (1991)). The EPA SW-846 Organic Methods Work Group have approved draft immunoassay methods for inclusion into the next revision of the SW-846 methods and for use in a variety of environmental testing applications. From this information it is obvious that newly developed reagents and immunoassays have become acceptable analytical tools for the environmental field.
Benzene is a carcinogenic environmental contaminant. Federal and state regulations have been promulgated to curb further benzene pollution and to aid in the remediation of previously contaminated sites. An effective immunoassay for the detection of benzene would dramatically assist in this effort by facilitating site characterization, remediation and monitoring activities.
The technical challenges associated with producing an immunoassay method for benzene, however, are significant. Benzene is an unremarkable monocyclic aromatic molecule that is structurally a component of many other compounds found commonly in nature. For example, the amino acid phenylalanine is a constituent of virtually all proteins and contains a phenyl group similar in conformation to benzene. An immunoassay that used an antibody that would bind to benzene would suffer from significant interference caused by the binding to structurally related compounds in the sample. The production of an antibody to benzene could also prove difficult since the stimulation of the immune response to benzene, and an antibody cross-reactive with phenylalanine may result in an autoimmune-induced pathogenic reaction within the host animal.
Peck, PCT Application WO 88/09798, discloses an immunoassay for aromatic ring containing compounds, including toluene, toluidine, benzene, styrene, etc. However there is no discussion of how to provide a monoclonal antibody which will detect the presence of benzene, without detecting toluene or other similar aromatic compounds.
Ligler et al., U.S. Pat. No. 5,183,740, disclose a method and apparatus for performing immunoassays. In particular, Ligler et al disclose several specific assays for detecting nitrated compounds such as dinitrophenol and trinitrotoluene, but no mention of an assay which allows detection of benzene, by way of nitrobenzene, without interference due to cross-reactivity with closely related compounds such as nitrotoluene.
Thus, an immunoassay method is needed which will provide reliable accurate and fast results in the field for detection of immunologically non-remarkable compounds, such as benzene. Such an assay would increase the efficiency of environmental site management activities such as characterization (mapping), remediation monitoring, and regulatory compliance.